There is growing evidence that several childhood solid tumors demonstrate the cytogenetic hallmarks of gene amplification in a significant percentage of cases. Identification of the putative amplified gene in these tumors could potentially lead to new understanding of the biology of these tumors, as well as their clinical behavior. Thus far, efforts to identify these genes have been limited primarily to screening tumor DNA with random oncogene probes by Southern blot hybridization. A more direct method would be to clone the amplified DNA in these tumors. The long term goal of this application is to clone amplified DNA in a two different pediatric malignancies that show evidence of gene amplification and subsequently Isolate a full length cDNA clone corresponding to the amplified gene. We plan to utilize the Roninson in-gel denaturation and renaturation technique, in concert with genomic subtraction libraries, to isolate and clone amplified DNA in a rhabdomyosarcoma cell line (Rh28) with an HSR. Subsequent efforts would be directed toward a hepatoblastoma (HB2) with DMs. Amplified genomic clones will be identified by slot blot, Southern blot , and in situ hybridization. In addition, the normal chromosomal location of amplified DNA will be determined by in situ hybridization. Transcribed genomic clones will then be identified by differential screening of the subtraction and Roninson libraries utilizing total tumor and representative normal cDNA as probes. Alternatively, DMs and HSRs will be isolated by physical separation techniques and subgenomic cosmid or lambda phage libraries will be constructed. Expressed DNA sequences would then be identified by screening the library with total tumor cDNA. Once an amplified and expressed genomic clone is identified, it will then be used as a probe to screen a tumor specific cDNA library. A full length cDNA clone(s) will then be isolated and sequenced in a standard fashion. Future studies would focus on screening a series of identical and similar malignancies with newly isolated cDNA probes by Southern blot hybridization to determine the frequency of gene amplification and identify any clinical correlations. A long term goal would be to initiate basic functional studies of any newly identified genes.